Throughout history, infectious diseases were a leading cause of human death. In the 20th century, social improvements, antimicrobial chemotherapy and immunization led to a brief period in which infectious diseases were viewed as torments of the past. However, the emergence of new infectious agents and the reemergence of old diseases demonstrated that continued awareness, research and development are necessary to limit the impact of infectious diseases on human health. Tuberculosis (TB) remains the second leading cause of human death from an infectious disease. Drug resistant strains of Mycobacterium tuberculosis (Mtb) threaten the success of TB control programs woridwide and new drugs are needed to effectively treat patients suffering from drug resistant TB and to prevent the spread of drug resistant TB. Genes Mtb requires for growth in vitro and during infections are among the most attractive targets for the development of new drugs. However, approximately a third of Mtb's in vitro essential genes remain of unknown function, which severely limits their value for drug development. The long term goal of this application is to overcome this limitation for drug development and to increase our understanding of the biological processes that are fundamental to the growth and survival of Mtb. To achieve this we will utilize conditional gene silencing approaches to construct conditional Mtb knockdown mutants that allow the partial inactivation of In vitro essential genes. We will then (i) perform extensive phenotypic characterization to better understand when and why a gene is required for growth and when and if its inactivation is lethal to the pathogen, and (ii) apply a variety of functional genomics approaches to mechanistically characterize gene functions.